The present invention relates generally to chemical compositions and methods for recognizing, attracting, adsorbing, isolating or selectively binding certain chemical compounds. More particularly, the present invention relates to the preparation of molecularly imprinted polymer (MIP) compositions and the use of such MIP compositions as selective adsorbents for peptides and other compounds that contain amino acid sequences having at least one N-terminal histidine residue or for histamine.
Molecular imprinting is a process for synthesizing materials that contain highly specific recognition sites for smaller molecules. Molecular imprinting techniques have generally been in use since the early 1970""s, when it was first demonstrated that a highly crosslinked organic network polymer could serve as a scaffold for the molecular imprinting of a template of an organic molecule. Today, molecular imprinting is used for many purposes, including the creation of macromolecular binding and catalytic sites and as binding sites for the separation or resolution of optical isomers or enantiomers. See, Hart, B. R., Rush, D. J. and Shea, K. J., Discrimination between Enantiomers of Structurally Related Molecules: Separation of Benzodiazepines by Molecularly Imprinted Polymers, J. Am. Chem. Soc. Vol. 122, pp. 460-465 (2000).
The preparation of a MIP generally involves polymerizing functional monomers and crosslinking agents in the presence of a xe2x80x9ctemplatexe2x80x9d compound. For example, a molecularly imprinted polymer may be prepared by polymerizing a mixture that contains a template compound, one or more polymerizable functional monomers, crosslinking agent(s), solvent(s), and a free radical initiator. Prior to polymerization, the monomer molecules distribute themselves around the template molecule in accordance with the size, polarity and functionality of the template molecule. As the polymerization reaction progresses, a rigid, three-dimensional polymer network is formed about the template. The template is removed, leaving an xe2x80x9cimprintxe2x80x9d or region in the polymer network that is complementary in size, shape, and functional group orientation to the template molecule.
More specifically, two general approaches have heretofore been developed for the preparation of MIPs. In one approach, the template is covalently bound to a polymerizable monomer and, after the polymerization has been completed, the covalent bond is cleaved so as to release the template from the polymer network. In the other approach, polymerizable monomers arrange themselves about a template based on noncovalent attractions or interactions (e.g., H-bonding, hydrophobic, ionic, steric or electrostatic interactions) and thereafter, after the polymerization has been completed, the non-covalently bound template is extracted, leached out or removed from the polymer network.
Molecular imprinting has proven to be useful in various applications, including chiral stationary phases (CSPs); as antibody mimics; as catalysts in organic synthesis; as mimics for particular enzymes and as biosensors in which the molecularly imprinted polymers are used as substitutes for other biological molecules.
In many of the methods previously known for preparation of MIPs, the monomers used have not been soluble in water or aqueous media at the concentrations utilized in the polymerization reaction. Since the use of an aqueous environment is desirable in many MIP preparations, such as where a MIP is prepared to be imprinted for a peptide or identifying segment of a peptide, there remains a need in the art for the development of new processes for aqueous-based preparation of MIPs.
The present invention provides methods for preparing molecularly imprinted polymer wherein the template comprises a peptide or amino acid sequence that has an N-terminal histidine residue or histamine. The method of the present invention generally comprises the steps of:
(A) providing a template complex that has the general formula 
wherein M is nickel, cobalt, copper, zinc, lead or manganese, P is a template peptide or amino acid sequence having at least one N-terminal histidine or a histamine molecule, n=0 through 10, X is NH, O, S or is absent and A is H or alkyl;
(B) polymerizing the template complex provided in Step A with at least a first monomer and optionally said first monomer and a second monomer, to form a polymer network containing the template complex;
the first monomer having the general formula 
wherein Y is NH, O or S or is absent, n1=0 through 10, n2=0 through 2, n3=0 through 10 and B is H or alkyl
and the second monomer, if used, having the general formula 
wherein Zxe2x95x90NH2, OH, Oxe2x80x94(CH2)n wherein n=2 through 4, NH(alkyl) or N(alkyl)2 and Q is H or alkyl.
After the polymerization is complete, the template complex may be removed from the polymer network, leaving the molecularly imprinted polymer network in tact.
Further in accordance with the present invention, the template complex, monomer(s) and reagents used in the above method may be soluble in water at the concentrations used in the method.
Still further in accordance with the present invention, the template complex provided in Step A may be prepared by combining a polymerizable methacrylamide-NTA-Ni2+ complex with a peptide or an amino acid sequence having at least one N-terminal histidine or histamine. The polymerizable methacrylamide-NTA-Ni2+ complex may be prepared by reacting NTA with NiSO4 or any other suitable Ni(II) source.
Still further in accordance with the invention, the first monomer used in Step B may comprise N,Nxe2x80x2-ethylene bisacrylamide or N,Nxe2x80x2-methylene bisacrylamide.
Still further in accordance with the present invention, the optional second monomer (if used in Step B) may comprise a water soluble acrylamide, such as acrylamide or methacrylamide.
Still further in accordance with the present invention, there are provided methods and compositions for adsorbing a target compound that contains a predetermined amino acid sequence having at least one N-terminal histidine. These methods generally comprise the steps of i) providing a molecularly imprinted polymer prepared according to the method set forth hereabove in this summary of the invention, wherein the amino acid sequence used to prepare the template complex in Step A is the predetermined amino acid sequence and ii) contacting the molecularly imprinted polymer with the material such that the target compound contained in the material will be adsorbed by the molecularly imprinted polymer.
Still further in accordance with the present invention, there are provided methods and compositions for determining whether a material contains a target compound that contains a predetermined amino acid sequence having at least one N-terminal histidine. These methods generally comprise the steps of i) providing a molecularly imprinted polymer that has been prepared according to the method of this invention as set forth hereabove in this summary of the invention, wherein the wherein the amino acid sequence used to prepare the template complex in Step A is the predetermined amino acid sequence, ii) contacting the molecularly imprinted polymer with the material such that any substantial quantity of target compound within the material will be adsorbed by the molecularly imprinted polymer and, thereafter, iii) determining whether the molecularly imprinted polymer has adsorbed any of the target compound.
Still further in accordance with the present invention, there are provided molecularly imprinted polymer compositions that have been prepared by the method of the present invention as set forth hereabove in this summary of the invention and examples of which are described in detail herebelow.
Still further aspects and advantages of the present invention will become apparent to those of skill in the art upon reading the detailed descriptions of examples and embodiments, set forth herebelow.